ABSTRACT Clonal hematopoiesis, frequently found in people over 70 years of age, occurs when a single hematopoietic stem/progenitor cell dominates all other stem cells in the bone marrow and yields a large fraction of all differentiated blood cells, and may aggravate coincident cardiovascular disease or progress to myeloid malignancy. Accumulation of genetic and epigenetic variability permits the cells to sample a diverse set of gene expression states allowing selection of hematopoietic clones with the highest fitness, especially after genotoxic exposures such as tobacco smoke or prior cancer therapy. Mutations in epigenetic modifiers such as DNMT3A are ancestral events occurring in age-related clonal hematopoiesis, and are enriched in pre-leukemic and leukemic HSCs that persist following chemotherapy. Preliminary studies in Dnmt3a-mutant knock-in mice that we developed show substantial functional variability among Dnmt3a-mutant hematopoietic clones compared to controls. Conventional ensemble methods mask cell-to-cell heterogeneity due to population averaging, while single-cell techniques are not amenable to prospective isolation of cells for functional assays. We developed an in vivo multicolor clonal tracking system, allowing easy characterization of clonal composition in the bone marrow over the lifespan of an animal, enabling capture of clonal evolution at unprecedented temporal resolution. Importantly, this system is suitable for isolation of individual hematopoietic clones by fluorescence-activated cell sorting, enabling functional and ?omics analyses. In this proposal, we track clonal architecture and emergence of dominant clones in vivo in real time, in steady-state and under selective pressures, and evaluate the contribution of specific genetic backgrounds such as presence of Dnmt3a mutations into this process. Next, we isolate dominant (?winners?) and non-dominant (?losers?) clones and perform transcriptomic, epigenomic, and chromatin profiling analyses, to investigate specific pathways and epigenetic state(s) driving clonal dominance. This will be done using conventional ensemble methods in FACS-sorted dominant clones (RNA-seq, DNA bisulfite sequencing, ATAC-seq), and complemented by MAPit technology allowing simultaneous analysis of DNA methylation and nucleosome positioning over long stretches of single molecules of DNA, to add granularity to the epigenome heterogeneity studies. These results are cross-validated by functional long-term self-renewal assays ex vivo in isolated dominant and non-dominant bone marrow cells, and by single-cell transcriptomic analysis. These findings will deepen our understanding of clonal evolution in the hematopoietic system. More importantly, the results of our studies will generate a rich source of potential therapeutic targets to hinder or prevent the progression to pre-malignant states and to frank leukemia. !